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Investigating the larvacidal activity of phytoecdysteroids found in Asian subspecies of Spinacia oleraca on
Tenebrio molitor
Zhao Xing Liang (4S2)Shi Guan Ming (4S1)
BackgroundMolting
• Process that arthropods undergo • The shedding and re-growth of a new
exoskeleton (The Insect Process of Molting, 2010)
Ecdysteroids
• Insect molting hormones• regulate growth, reproduction, and
development (Grebenok, Galbraith, Benveniste, Feyereisen, 1996)
Phyto-ecdysteroids •Exact replica of ecdystroids produced by plants•Protective mechanism•Disrupt development of insects
Background
- Spinacia oleracea (Spinach)
- Chenopodiaceae family - Produces ecdysteroids
structurally similar to those produced by
arthropods
- Tenebrio molitor (yellow mealworm)- Order Coleoptera- Viewed as pests in
countries such as Mexico (University of California,
2009).
Objectives
Find the most
effective way in
extracting phytoecdy
steroids from
Spinacia oleracea
Investigate the
larvicidal activity of
phytoecdysteroids by
using Tenebrio
molitor as an
indicator
Develop a novel
insecticide that is both
effective and
environmentally
friendly
Rationale
Larvicide
Effective
Biodegradable, environmentally friendly
Phytoecdysteroids are beneficial to human
health (Mária Báthori et al, 2005).
Some larvae have acquired resistance against conventional
methods (Rangel et al, 2009).
So Therefore
could be used to exterminate
mealworm larvae in food items
Hypothesis
• The larvae of Tenebrio molitor, exposed to phytoecdysteroids extracted from Spinacia oleracea, will have a higher rate of mortality and deformities.
Variables
Controlled Independent Dependent
• Species of mealworm larvae
• Culture conditions
• Extraction Method
•Concentration of extracts
• Mortality Rate of T. molitor
• Observable deformities of T.
molitor
Materials and apparatus for extraction of Phytoecdysteroids
Materials• Methanol• Hexane• Dichloromethane• Acetone• Ethanol of 96% purity• Alumina• Octyl silane• Cotton wool
Apparatus• Vacuum pump• Column
Materials and apparatus for Bioassay on Tenebrio Molitor
Materials• Boxes• Mealworm feed• Cotton wool• Petri dishes
Apparatus• Spraying apparatus• Micro syringe
Overview of MethodologyPurchase,
cutting and drying of Spinacia oleracea
Fractionated precipitation
Solvent-solvent distribution
Column chromatographyHPLC
Reverse phase chromatography
Bioassay
Set up for BioassayConcentration
of phyto-ecdysteroids
Method foradministration Number of Meal worms
Solvent Control Petri Dish 1 Petri Dish 2 Petri Dish 3
1
Injection 10 10 10 10
Spraying 10 10 10 10
Ingestion 10 10 10 10
2
Injection 10 10 10 10
Spraying 10 10 10 10
Ingestion 10 10 10 10
3
Injection 10 10 10 10
Spraying 10 10 10 10
Ingestion 10 10 10 10Negative
Control N/A 10
References• Adler, J. H., Grebenok, R. J. (1999). Occurrence, biosynthesis, and putative role of ecdysteroids in plants.
Critical Reviews in Biochemistry and Molecular Biology, 34(4), 253-264.
• Bakrim, A., Maria, A., Sayah, F., Lafont, R., Takvorian, N. (2008). Ecdysteroids in spinach (Spinacia oleracea L.): Biosynthesis, transport and regulation of levels. Plant Physiology and Biochemistry, 844-854.
• Grebenok, R.J., Galbraith, D.W., Benveniste, I., & Feyereisen, R. (1996). Ecdysone 20-monooxygenase, a cytochrome p450 enzyme from spinach, Spinacia oleracea. Phytochemistry, 42(4), 927-933.The Insect Process of Molting. (2010). Retrieved from http://www.insectidentification.org
• Malausa, T., Salles M., Marquet V., Guillemaud T., Alla, S., Marion-Poll, F., Lapchin L. (2006). Within-species variability of the response to 20-hydroxyecdysone in peach-potato aphid (Myzus persicae sulzer), Phytochemistry, 52, 480-486.
• Savolainen, V., Wuest, J., Lafont, R., Connat, J. L. (1995). Effects of ingested phytoecdysteroids in the female soft tick Ornithodoros moubata. Phytochemistry. 51, 596-600.
• Schmelz, E. A., Grebenok, R. J., Ohnmeiss, T. E., Bowers, W. S. (2002). Interactions between Spinacia oleracea and Bradysia impatiens: a role for phytoecdysteroids. Archives of Insect Biochemistry and Physiology, 51, (204- 221).
• University of Arizona. (1997). Darkling Beetle/Mealworm Information. Retrieved from September 26, 2010 http://insected.arizona.edu/mealinfo.htm
• University of California (2009). Mealworms and Darkling Beetles (Tenebrio beetle). Retrieved September 26, 2010 from http://lhsfoss.org/fossweb/teachers/materials/plantanimal/tenebriobeetles.html
Thank YouQuestions?
Detailed Methodology
Methodology (Extraction)
• Fractionated precipitation• Dried plant of 6g is extracted with Methanol at a mass-volume ratio of
1:10 ( 60ml methanol needed)• After extraction, the methanolic solution is split into 3 parts. (20 – 21 ml
each)• The first part of the solution (20ml) is mixed with half the volume of
acetone (11ml) while the second part (20ml) is mixed with same volume of acetone (22ml) and the last part is mixed with twice the volume of acetone (40ml).
• The resulting solution is then filtered and the residue is removed.• The residue is washed with the same ratio of methanol and acetone as
step 1 and 3 respectively • The washing solution is then added to the filtrate. • The solutions are then evaporated.• The crude extracts are redissolved in methanol at the same mass-volume
ratio of 1:10.• Step 2-8 is repeated 2 more times.
Methodology (Extraction)• Solvent-solvent distribution• After precipitation, the crude extracts are dissolved
in 50% aqueous methanol (specific numbers needed – add until everything dissolves)
• Hexane (how much?) is added to the solution to extract the non-polar compounds in the precipitate.
• The aqueous methanol phase (bottom) is separated (using separating funnel) and then evaporated to dryness.
• The resulting residue is dissolved in pure methanol.• The methanolic solution is mixed with aluminium
oxide and the suspension was evaporated to dryness with a rotary evaporator.
Methodology (Extraction)• Chromatography• The alumina is eluted with a hybrid of Dichloromethane- 96%
Ethanol solution of ratio 9:1 and 8:2. (need to do 2 times)• A cotton wool of mass of 0.2 g was placed at the bottom of
the column to prevent alumina from flowing out.• 70-90g (subject to experimental changes) of Alumina is mixed
with the eluent.• The mixture of alumina and eluent was stirred and poured
into the column until is 75% full.• The bands in the mobile phases are collected in different
beakers for further tests.
Methodology (Extraction))• Purification of Ecdysteroids• For further purification, the ecdysteriods are separated by reversed-phase
chromatography• In reversed-phase chromatography, octyl silane is used as the stationary
phase (being non-polar) instead of silica/alumina.• A cotton wool of mass of 0.2 g was placed at the bottom of the column to
prevent alumina from flowing out.• 70- 90g (subject to experimental changes) Octyl Silane is mixed with the
eluent. • To control the flow of the mobile phase, a vacuum will be used at the
outlet. • Different concentrations of methanol are used as eluents in this
chromatography.• Stepwise gradient elution is used with an increase of 5% of methanol
content in each step.• Different bands of ecdysteroids will be formed on the stationary phase.• The specific hormone, 20-hydroxyecdysone is isolated with methanol of
35-40% purity.
Methodology (Extraction)
• Preparation of phytoecdysteroids for bioassay
• Evaporate the solution to dryness• Dissolve the phytoecdysteroids in water to the
desired concentration
Methodology (Bio-Assay)
1. 10 last-instar mealworm larvae are placed in a box per setup.
2. A determined amount of extracts are sprayed onto the mealworms.
3. The mealworm larvae are left to develop for 30 days.4. The deformities and mortality rate of the mealworm
larvae is recorded after a day, 15 days and 30 days.5. Step 1 to 4 is repeated with the extracts being injected
or fed to the mealworm larvae.